In 2008 more than 200,000 American women will be diagnosed with breast cancer and many will be treated with antiestrogens. Unfortunately, many breast tumors initially respond to antiestrogens will become unresponsive over time, leading to metastasis and mortality. The molecular mechanisms of breast cancer progression are largely unknown, which hinders the development of targeted therapies for advanced cancer. Our recent studies showed that ID1, an inhibitor of differentiation, played a role in promoting breast cancer cells to adopt an aggressive, hormone-independent phenotype, resulting in antiestrogen resistance. The significance of our finding is underscored by recent reports that ID1 is highly expressed in poorly differentiated, metastatic tumors. The primary function of ID1 is to inhibit DNA binding of a family of transcription factors that contain a basic helix-loop-helix (bHLH) domain. To identify the potential bHLH transcription factor that is regulated by ID1 in breast cancer cells, we performed a comprehensive bioinformatic analysis of published gene expression data of human breast tumors. Meta-analysis revealed that the expression of BHLHB2, a bHLH transcription factor and putative target of ID1, is strongly associated with hormone-dependent breast tumors, and BHLHB2-coexpressed genes significantly overlap with genes coexpressed with ESR1, FOXA1 and GATA3, the key transcription factors that specify the hormone-dependent phenotype of breast cancer. In addition, we found that E-box motif, the cis-regulatory sequence recognized by BHLHB2, was overrepresented in ESR1 direct target genes. We hypothesize that BHLHB2 is an integral component of a transcription regulatory network that dictates the hormone-dependent phenotype of breast cancer, and ID1 facilitates tumor cells to acquire a poorly differentiated phenotype by inhibiting BHLHB2 function. We will test this hypothesis in the following three specific aims by conducting a systematic and detailed study on the function and regulation of BHLHB2 in breast cancer cells. Specifically, we will: 1) investigate the role of BHLHB2 in maintaining hormone-dependent phenotype and its regulation by ID1 by examining the consequences of gain- or loss-of- function of BHLHB2 and ID1 in human breast cancer cells; 2) identify and characterize genes regulated by BHLHB2 and ID1 using high-throughput genome-wide analysis of gene expression and BHLHB2 DNA binding; and 3) investigate the regulation and function of fulvestrant resistance-related miRNAs, with emphasis on the cross-regulation between the fulvestrant resistant-relate miRNA and core transcription factors of hormone- dependent breast cancer cells.